14-3-3 proteins在細胞中扮演相當重要的角色，影響許多細胞的作用機制包括：細胞週期的進行、細胞凋亡及細胞內訊號傳遞等。人類的14-3-3 proteins至少包含有7種 isoforms，其中的14-3-3 gamma不僅在腦部含量豐富，而且表現在許多神經系統疾病患者的腦脊液中。儘管14-3-3 gamma在腦部具有重要生理功能，但是目前尚無14-3-3 gamma在腦瘤形成機制中所扮演角色之報告。因此，此本研究利用即時定量聚合酵素連鎖反應、反轉錄聚合酵素連鎖反應、西方墨點法、免疫組織染色及等技術，對三種腦瘤細胞株T98G、U87-MG、U251及4個正常腦部組織、24個星狀細胞瘤、14個神經膠母細胞瘤、2個寡樹突膠質細胞瘤、1個室管膜瘤檢體進行14-3-3 gamma基因及其表現之檢測。實驗結果發現：在三種腦瘤細胞株中，14-3-3 gamma mRNA表現比正常腦部組織高。但是14-3-3 gamma 蛋白質表現則比正常腦部組織低。在腦瘤組織方面，24個星狀細胞瘤檢體中，14-3-3 gamma mRNA的表現比正常的腦部組織多的有20個(83%)，14-3-3 gamma 蛋白質的表現比正常的腦部組織多的有9個(37%)。全部14個神經膠母細胞瘤檢體，14-3-3 gamma mRNA的表現均比正常的腦部組織高(100%)，14-3-3 gamma 蛋白質的表現比正常的腦部組織高的有9個(64.3%)。同時也發現在神經膠母細胞瘤中14-3-3 gamma 蛋白質表現量比星狀細胞瘤高出許多。至於星狀細胞瘤及神經膠母細胞瘤檢體的基因組DNA上基因數copy number均比正常腦組織高。因此，由本研究結果顯示14-3-3 gamma可能在惡性腫瘤生成的過程中扮演重要角色。

The family of 14-3-3 proteins is crucial for various physiological cellular processes such as signaling, cell growth, division, differentiation, and apoptosis. One of the 14-3-3 proteins members, 14-3-3 gamma, is abundantly expressed in brain and had been detected in the cerebrospinal fluid of
patients with different neurological disorders. Although 14-3-3 gamma played critical physiological or pathological role in brain, it has not been reported on brain tumorigenesis. To test expression of 14-3-3 gamma in brain tumor, 3 brain tumor cell lines and 4 normal brain tissues, 24 astrocytoma, 14 glioblastoma mutiform, 2 oligodenroglioma, 1 ependymoma were analyzed using RT-PCR, western blotting, immunohistochemistry, real-time quantitative PCR. The study found that the expressions of 14-3-3 gamma mRNA in all of tumor three cell lines was greater than normal brain tissue, but the 14-3-3 gamma proteins expressed were lower than normal brain tissue. In brain tumor tissues, higher 14-3-3 gamma mRNA expression was detected in 20 of 24 astrocytoma (83%) and higher 14-3-3 gamma protein expression was detected in 9 of 24 astrocytoma (37%). The expression of 14-3-3 gamma mRNA is higher than normal brain tissue in all 14 glioblastoma multiforme (100%), and the 14-3-3 gamma protein was expressed higher in 9 of 14 glioblastoma multiforme than normal brain tissue (64.3%). Besides, the 14-3-3 gamma protein expressed much higher in glioblastoma multiforme than astrocytoma .The copy number of the 14-3-3 gamma gene was higher in astrocytoma and glioblastoma multiforme than normal brain tissue. Thus, this study evidenced that the 14-3-3 gamma protein may play a crucial role during tumorigenesis of brain tumors.

目 錄
壹、緒論 …………………………………………………p.1
一、前言 …………………………………………………p.1
二、腦瘤 …………………………………………………p.2
三、14-3-3 proteins……………………………………p.6
貳、研究目的 ……………………………………………p.10
參、材料與方法 …………………………………………p.11
一、細胞 …………………………………………………p.11
二、檢體 …………………………………………………p.11
三、反轉錄聚合酵素連鎖反應(Reverse transcription-
polymerase chain reaction;RT-PCR) ………………p.11
四、西方墨點法（Western Blot Analysis）…………p.13
五、免疫組織染色(Immunohistochemistry) …………p.16
六、Real-time quantitative PCR ……………………p.17
七、定序 …………………………………………………p.18
肆、結果 …………………………………………………p.20
一、14-3-3 gamma在正常腦部組織的表現 ……………p.20
二、14-3-3 gamma 在腦瘤細胞株的表現………………p.20
三、14-3-3 gamma 在腦瘤組織的表現…………………p.20
四、14-3-3 gamma gene copy number…………………p.21
五、定序結果 …………………………………………p.21
六、免疫組織染色結果 …………………………………p.22
伍、討論 …………………………………………………p.23
陸、參考文獻 ……………………………………………p.24
柒、圖表 …………………………………………………p.35
圖 表 目 錄
表一 人類的14-3-3 isoforms 及其特異性 ………… p.35
圖一 14-3-3 proteins的作用模式圖 …………………p.36
圖二 14-3-3 proteins在細胞週期的角色 ……………p.37
圖三 14-3-3 proteins在apoptosis的角色……………p.38
圖四 14-3-3 proteins在細胞信號傳遞的角色 ………p.39
圖五 正常腦部組織中14-3-3 gamma mRNA及蛋白質的
表現 ……………………………………………… p.40
圖六 腦瘤細胞株中14-3-3 gamma mRNA及蛋白質的表現p.41
圖七 腦瘤組織中14-3-3 gamma mRNA的表現 …………p.42
圖八 腦瘤組織中14-3-3 gamma 蛋白質的表現 ………p.43
圖九 腦瘤組織中14-3-3 gamma mRNA及蛋白質表現之定量結
果 ……………………………………………p.44
圖十 腦瘤及正常腦部組織中14-3-3 gamma gene copy
number之比較………………………………………p.46
圖十一 14-3-3 gamma mRNA及蛋白質在astrocytoma 及
glioblastoma multiforme中的表現……………p.47
圖十二 定序結果…………………………………………p.48
圖十三 免疫組織染色結果-glioblastoma multiforme
…………………………………………………p.49

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